Investigation into the genetic basis of bovine horn development

Abstract

The presence of horns in ruminants has financial and welfare implications for the farming of cattle, sheep and goats worldwide. The genetic interactions that lead to horn development are not known. Hornless, or polled, cattle occur naturally, but the known causative DNA variants (Celtic, Friesian, Mongolian and Guarani) are in intergenic regions on bovine chromosome 1, and therefore, their functions are not known. The leading hypothesis is that horns are derived from cranial neural crest cells and the POLLED variants disrupt the migration or proliferation of these stem cells. The bovine POLLED region was explored through bioinformatics analyses as horned animals may have genomic differences from hornless individuals or species near the POLLED DNA variants. The aim was to identify differences in genes synteny, lincRNA, and topologically associating domain (TAD) structure between horned and hornless individuals or species. Horned (n = 1) and polled (Celtic; n = 1) Hi-C sequences produced the same TAD structures. The POLLED genomic region was refined to a 520-kb region encompassing all four POLLED variants. LOC526226 was unique to the bovine POLLED region and not conserved in the species analysed (water buffalo, sheep, goat, pig, horse, dog and human), and therefore, may be involved in horn development. Histological analyses of cranial tissues from homozygous horned and polled fetuses at day 58 of development were conducted. The aims were to 1) determine the differences in the structure of horn bud region, and 2) compare immunohistochemistry staining of neural crest markers (SOX10 and NGFR) and RXFP2 between horned and polled tissues. Condensed cells were only observed in the horn bud mesenchyme of horned fetuses and may be progenitor cells. SOX10 and NGFR was not detected in these condensed cells, and therefore, these cells are not derived from the neural crest or have differentiated and no longer express neural crest markers. SOX10 and NGFR were detected in the peripheral nerves. RXFP2 was detected in peripheral nerves and in the horn bud epidermis. Transcriptomic analyses of cranial tissues from the horned and polled fetuses at day 58 of development was also conducted. The aims were to 1) identify genes that may directly be affected by the polled variants, and 2) identify genes and pathways important for horn development. Near the POLLED region, three genes (C1H21orf62, SON and EVA1C) and one lincRNA (LOC112447120) were differentially expressed between horned and polled fetuses. Previously identified candidate genes, RXFP2, TWIST2 and ZEB2, were also differentially expressed. New candidates for the horn development pathway were proposed based on the analyses (MEIS2, PBX3, FZD8, CTNNB1 and LEF1). LOC526226 was not differentially expressed in the horn bud. Differentially expressed genes had functions in axon guidance, cytoskeletal structure and the extracellular region, and therefore, these pathways may be vital for horn development. Based on this research, it is now hypothesised that 1) horn stem cells are located in the mesenchyme and interact with the epidermis to initiate horn development, 2) the Celtic POLLED variant directly affects expression of C1H21orf62, SON, EVA1C and LOC112447120, and 3) the migration of horn stem cells is reduced by the effect of the POLLED variants upon C1H21orf62, SON, EVA1C and/or LOC112447120 expression.